Proteomics and metabolomics combined study on endopathic changes of water‐soluble precursors in Tan lamb during postmortem aging

Abstract Tan lamb is highly recommended breed in China. It is of great significance to understand the underlying mechanism of how water‐soluble flavor precursors metabolize in Tan lamb muscles during the postmortem aging period. In this study, we investigated the muscle pH, lactate dehydrogenase (LDH) activity, and the variations in water‐soluble flavor‐related metabolites. The proteome changes were profiled to provide insights into the biochemical changes affecting accumulation of water‐soluble flavor precursors in different aging stages (days 0, 4, and 8). The results indicated that pH value considerably decreased from day 0 to day 4, and increased from day 4 to day 8 (p < .05). The activity of LDH significantly increased from day 0 to day 4, and decreased from day 4 to day 8 (p < .05). Postmortem glycolysis was activated in 4 days, which directly affected the variations in metabolic enzymes and triggered the accumulation of flavor‐related carbohydrates. The free amino acids accumulated due to hydrolysis of structural proteins, with 3‐hydroxy‐L‐proline, aspartic acid, and methionine increasing from day 0 to day 4, and aspartic acid, serine, threonine, tyrosine, phenylalanine, and D‐phenylalanine from day 4 to day 8. The inosine and hypoxanthine accumulated due to the degradation of ATP. The results of the present study provide insightful information, revealing the differences in biochemical attributes in Tan lamb muscles caused by postmortem aging.

MS-based metabolomics was used to investigate the effects of castration on sheep muscles, the result of which highlighted that lipids and hydrophilic metabolites were affected by the castration, contributing partly to the improvement of the quality of mutton (Li et al., 2020).
When it comes to Tan lamb, no data are available on the integrated application of proteomics and metabolomics to characterize the effects of postmortem aging on the water-soluble flavor precursors. In this study, the muscle pH and lactate dehydrogenase activity in postmortem Tan lamb muscles were investigated, and the proteome and metabolite profiles were examined using proteomic and metabolomics approaches. This study aimed to obtain more insights into the potential causes for the formation of water-soluble flavor precursors during postmortem aging.

| Preparation of animals and samples
All the Tan lambs used for the study were grazed and raised under the same conditions. Eight Tan lambs (male, approximately 6 months old) were randomly selected and humanely slaughtered at a commercial meat company (Daxia Food Co. Ltd., Yanchi, Yinchuan, China) following Islamic practice and requirements of National Standards of PR China (GB 12,694-2016) without applying electric stimulation. The longissimus dorsi muscles were cut off immediately after slaughter, transferred to a polystyrene tray sealed with polyethylene films, and stored for 8 days (4ºC, relative humidity of 80%). A sterilized knife was used to collect samples (6 g) from the inside of longissimus dorsi at days 0, 4, and 8. The collected samples were immediately placed in a liquid nitrogen tank and transferred to a refrigerator at −80 ºC for further analysis. All the experiments were repeated with three independent biological replicates. The result of day 4 versus day 0 was recorded as Group 4/0, and day 8 versus day 4 as Group 8/4.

| Detection of muscle pH
According to the method of Al-Dalali et al. (2022), muscle pH was measured using a portable pH meter (Youke P611,Shanghai,China) after preparing a mixture of 5 g sample and 45 ml of distilled water (homogenized Jingxin LC-11L, Shanghai, China) for 3 min. The pH meter was calibrated at pH 4.0, pH 6.86, and pH 9.18 (at 20°C) before the measurement of the samples.

| Determination of lactate dehydrogenase (LDH) activity
LDH activities were surveyed with Elisa Kit (Fankew Industrial Co.,

Ltd, Shanghai, China) according to the manufacturer's instructions.
Minced muscle (1 g) was lysed in 9 ml of phosphate-buffered saline (PBS, pH 7.2, 0.01 mol/L) using a homogenizer (Jingxin JXFSTPRP-24, Shanghai, China) at 4°C, and then centrifuged at 5,000 rpm at 4°C for 15 min, with the supernatant collected and the conjugate reagent (polygalacturonase, pectin methylesterase) serially diluted and added into a 96-well microtiter plate. The supernatant was incubated at 37°C for 30 min with conjugate reagent (50 µl), joined by chromogenic solution A (50 µl, sodium acetate, citric acid, 30% H 2 O 2 ) and B (50 µl, EDTA-Na, citric acid, glycerinum, tetramethylbenzidine), and incubated at 37°C in the dark for 15 min before adding the stop buffer (50 µl, H 2 SO 4 , 1 mol/L). The enzymatic activity was obtained by testing the absorbance at 450 nm using a Microplate reader (LabSystem Multiskan, Finland). The serial dilution method was applied to dilute the conjugate reagent with PBS (0.01 mol/L) to draw the standard curve, and the LDH activities were calculated according to the standard curve.

| Proteomic profiling of Tan lamb muscles by isobaric tags for relative and absolute quantitation (iTRAQ)
Each sample was accurately weighted for 5 g and transferred to a 1.5-mL centrifuge tube with 500 µl of sample lysate and a certain amount of protease inhibitor phenylmethanesulfonyl fluoride (PMSF) added to reach the final concentration of 1 mM. The muscle was homogenized and smashed by ice ultrasonic crushing for 3 min (twice, 40 kHz, 80 W, VCX130, Sonics, USA). The supernatant was collected after centrifugation (twice, 15,000 g, 15 min, Eppendorf, Germany) and mixed uniformly with 5-fold precooled acetone. After being placed at −20°C overnight, the precipitation mixed well with chilled acetone was centrifuged (3 times, 4°C, 12,000 g, 15 min), dried at 25°C, and dissolved in the sample lysis solution (3 hr). The protein solution was obtained after centrifugation (twice, 25°C, 12,000 g, 10 min). Protein concentration was determined with the BCA method (Smith et al., 1985).
Protein digestion was performed according to the method of Wiśniewski et al. (2009). Following the digestion, a C18 columns and a vacuum concentration meter (Thermo, USA) were used to desalt and dry the peptides. The peptides labeling was conducted then by re-dissolving the dried peptides powder for 100 μl with 100 mM TEAB. The samples were labeled with 8 plex-iTRAQ reagents (ABSCIEX, USA) as per the manufacturer's instructions. A Triple TOF 5600 Mass Spectrometer (SCIEX, USA) equipped with a Nanospray III source (SCIEX, USA) was utilized for proteomic profiling. Samples were loaded by a capillary C18 trap column (3 cm×100 µm, 3 µm, 150 Å, Eksigent) and then separated by a C18 column (15 cm×75 µm, 3 µm, 120 Å, Eksigent). The flow rate was 300 µl/min and the linear gradient was 90 min (from 5%-85% B over 67 min; mobile phase A = 2% ACN/0.1% FA and B = 95% ACN/0.1% FA).
Analyst TS 1.1 software (Thermo Fisher Scientific, Waltham MA, USA) was used to analyze the raw data of proteomics. Protein pilot 5.0 software (Ovis aries. fasta) was applied to identify and quantify proteins. Those with FDR ≤1%, PSMs <40%, and protein score ≥1.3 were selected as highly reliable proteins, and proteins were considered as differentially abundant proteins with a fold change (FC) ≥ 2 or ≤0.5 and p < .05.

| Identification of changes in watersoluble flavor precursors of Tan lamb meat during postmortem aging
The sample derivation was completed following the experimental protocols of You and Luo (2019). Each sample (30 mg) was transferred to a 1.5-mL Eppendorf tube, treated with 2-chloro-l-phenylalanine (20 μl,0.3 mg/ml,Hengbai Biotechnology Co.,Ltd.,Shanghai,China) in methanol (CNW Technologies GmbH, Düsseldorf, Germany), and ground (60 HZ, 120 s) after being stored at −80 ºC for 2 min, and added with 120 μl of chloroform. Following an ultrasonicassociated extraction (25°C, 10 min), the samples were stored at 4 ºC for 10 min, and centrifuged at 12,000 rpm at 4 ºC for 10 min.
Quality control samples were made by mixing aliquots of all samples to form pooled samples. An aliquot of 300 μl of supernatant was transferred to a glass sampling vial, vacuum dried at room tem- NIST 11 and Fiehn database were used to identify and quantify the differential metabolites. The data were analyzed using SIMCA software package (14.0, Umetrics, Umea, Sweden). The Hotelling's T2 region, shown as an ellipse in score plots of the models, defined the 95% confidence interval of the modeled variation. Variable importance in the projection (VIP) ranked the overall contribution of each variable to the OPLS-DA model and the variables with "VIP >1.00" and "p < .05" were considered relevant for group discrimination.

| Bioinformatic analysis
Gene Ontology (GO) annotation of differentially abundant protein was performed using OmicsBean platform (http://www.omics bean. cn/). The pathway analysis of differentially abundant protein and differential metabolite was queried against Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg/).

| Integrative analysis
The KEGG pathways were selected as the carrier to map the differential proteins and differential metabolites to elucidate the effects of postmortem aging on meat flavor precursors, only the pathways with p < .05 were considered to have significant enrichment. Metabolite-protein interactions control a variety of cellular processes, to further elucidate the internal relationships between differentially abundant proteins and differential metabolites. The Pearson correlation coefficient was calculated according to the expression or relative content, with the interaction network built using R software (version 3.6.3).

| Statistical analysis
The mean of the three samples was recorded and expressed in the form of the mean ±standard deviation (SD). The data were analyzed by IBM SPSS 26.0 (SPSS Inc., Chicago, IL, USA). A one-way analysis of variance (ANOVA) was used to determine statistical significance, where p < .05 was considered as significant difference.

| Muscle pH and LDH activity
The pH value of Tan lamb muscles conditioned at 4 ºC considerably decreased in the first 4 days (p < .05) and increased thereafter (p < .05). The minimum value was observed at day 4 and the maximum value immediately after slaughter ( Figure 1). Figure 2, LDH activity of Tan lamb muscles significantly increased from day 0 to day 4 postmortem with the maximum value recorded at day 4 (21.45 IU/L), and decreased from 21.45 IU/L to 16.89 IU/L after day 4 (p < .05) with the minimum value recorded at day 8 (16.89 IU/L).

| Protein profiles of Tan lamb muscles at different aging stages
The identification and quantitation of differentially abundant proteins of Tan lamb muscles aged for days 0, 4, and 8 at 4 ºC were performed with iTRAQ-based proteomics. The results showed that there were 29 and 52 differentially abundant proteins in Group 4/0 and Group 8/4, respectively, mainly metabolic enzymes, structural proteins, transporter proteins, stress proteins, and others (Table 1).
From day 0 to day 4 postmortem, there were 19 up-regulated and 10 down-regulated differentially abundant proteins. From day 4 to day 8 postmortem, there were 5 up-regulated and 47 down-regulated differentially abundant proteins (Figure 3a,b). Among the metabolic enzymes, glycogen phosphorylase, enolase, nucleoredoxin, and aconitase 1 were up-regulated from day 0 to day 4, and cytochrome c oxidase subunit 2 from day 4 to day 8.
The functional analysis of differentially abundant proteins was preformed through GO and KEGG annotation. The results of GO annotation showed that from day 0 to day 4, the identified proteins in Tan lamb responded to muscle contraction, muscle system process, and multicellular organismal movement in terms of biological process, served as structural proteins in terms of cell component, and functioned in binding, catalytic activity, and structural molecular activity in terms of molecular function. From day 4 to day 8, the identified proteins in Tan lamb responded for muscle contraction, muscle movement, and catabolic process in terms of biological process, served as structural proteins and microparticle in terms of cell component, and functioned in binding, catalytic activity, and structural molecular activity in terms of molecular function (Figure 3c,d). The results of KEGG pathway annotation showed that the differentially abundant proteins were involved in metabolism from day 0 to day 4, and in metabolism, environmental information processing, and organismal systems, including carbohydrate metabolism, amino acids metabolism, and signaling pathway from day 4 to day 8 (Figure 3e,f).
The differentially abundant proteins relating to Tan lamb flavor precursors are listed in Table 2. These proteins are mainly structural proteins, metabolic enzymes, and their co-factors, such as troponin, actin, tropomyosin, myosin light chain, myosin heavy chain, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, pyruvate kinase, and UDP-glucose pyrophosphorylase 2.

| Identification of differential metabolites of Tan lamb at different aging stages
The identification and quantitation of differential metabolites of muscles aged for 0, 4, and 8 days at 4 ºC were performed by F I G U R E 1 Changes in pH value of Tan lamb muscles. Note: Data are expressed in the form of mean ± SD (n = 3), a-b referring to significant difference (p < .05) F I G U R E 2 Changes in LDH activities on Tan lamb muscles. Note: Data are expressed in the form of mean ± SD (n = 3), a-c referring to significant difference (p < .05)  GC×GC-TOF-MS. The results showed that there were 22 and 21 target differential metabolites in Group 4/0 and Group 8/4, respectively. The differential metabolites were mainly the degradation products of nucleic acid, carbohydrates, free amino acids, and their derivatives ( Table 3).
The results from KEGG pathway annotation of target differential metabolites showed that the differential metabolites were mainly involved in carbon metabolism, citrate cycle (TCA cycle), glycolysis/ gluconeogenesis, pentose phosphate pathway, aminoacyl-tRNA biosynthesis, and amino acids metabolism from day 0 to day 4 and in purine metabolism, amino acids metabolism, biosynthesis, and degradation from day 4 to day 8 (Table 4).

| Joint analysis of proteomics and metabolomics
To elucidate the effects of postmortem aging on water-soluble flavor precursors of Tan lamb meat, the KEGG pathways were selected as the carrier to map the differentially abundant proteins and   day 8 (p < .05), suggesting that under postmortem anaerobic conditions, energy metabolism turned to anoxic degradation, LDH was activated, and subsequently glycogen was converted into lactic acid.
This was one of the reasons why muscle pH decreased before day 4. Numerous studies have revealed that hypoxic ischemia leads to increased activity of glycolytic enzymes in muscle cells at the early postmortem stages, and subsequently promotes the consumption of glycogen, decreases muscle pH, and accelerates the aging of muscle (Apaoblaza et al., 2015;England et al., 2015;Hardie, 2011;Lametsch et al., 2002). The result of the present study is consistent with the previous studies.
Pyruvate, one of the important metabolites of glycolysis, decreased before day 4 postmortem, probably due to rapid/excessive glycolysis converting pyruvate to lactic acid (Table 3). Pyruvate kinase increased from day 0 to day 4 (FC =1.49, Table 2), while higher pyruvate kinase concentrations and activities are hallmarks of a greater glycolytic metabolism in the muscle tissue (Baldassini et al., 2015). All of these data indicate that glycolysis was activated in Tan lamb muscles within 4 days postmortem. The concentration of enolase 3 was found to increase before day 4 ( Table 1). The upregulation of enolase 3 during postmortem storage was consistent with Holstein cattle semitendinosus muscle in which there was an increase of this enzyme after 4 days of aging (Yu et al., 2017).
Furthermore, triosephosphate isomerase was found to increase before day 4 and decrease from day 4 to day 8 (  (Wu et al., 2015). The report of Zuo et al. high-lighted that triosephosphate isomerase may have the potential to serve as biological markers for water-holding capacity prediction of yak meat (Zuo et al., 2016). Therefore, glycolysis enzymes contribute to the accumulation of flavor-related carbohydrates, and simultaneously, affect the development of other quality traits of meat.
Within 4 days of aging, the content of fructose-6-phosphate and glucose-6-phosphate increased, which could be due to the activation of glycolysis (Table 3) From day 4 to day 8 postmortem, fructose-bisphosphate aldolase, glucose-6-phosphate isomerase, triosephosphate isomerase, enolase 3, glyceraldehyde-3-phosphate dehydrogenase, fructosebisphosphatase 2, L-lactate dehydrogenase, phosphoglucomutase 1, phosphoglycerate mutase, phosphoglycerate kinase, and pyruvate kinase decreased (Tables 1 and 2), which could be attributable to the gradual degradation of the proteins in Tan lamb muscles with the extension of postmortem time. The decrease of fructose-bisphosphate aldolase, phosphoglycerate mutase, phosphoglucomutase 1, phosphoglycerate kinase, and enolase 3 during postmortem storage was consistent with that in Hengshan goat longissimus lumborum muscles stored at −18°C for 30 days .
The large number of nonvolatile compounds produced by glycolysis metabolites contribute to meat flavor upon heating, under the domination of the key enzymes of glycolysis, which is consistent with the result of Toldrá and Flores (2000). Glycolysis in postmortem Tan lamb muscles directly affecting variations in metabolic enzymes triggers the accumulation of flavor-related carbohydrates.

| Degradation of structural proteins leading to accumulation of amino acids
The compositions and contents of amino acids in meat are one of the criteria for assessment of nutritional value, and one of the most   cells leads to calcium homeostasis imbalance (Sierra & Oliván, 2013), and subsequently the activation of calpain-1 and caspase-3 to hydrolyze the myofibrillar proteins (Ahmed et al., 2015;Huang et al., 2009). Therefore, the content of metabolites such as 3-hydroxy-L-proline, aspartic acid, and methionine increased from day 0 to day 4, and aspartic acid, serine, threonine, tyrosine,  (Lee et al., 2016;Mottram, 1998). Research highlights that cystine, cysteine, and methionine can be decomposed to produce hydrogen sulfide, methyl mercaptan, and thioformaldehyde, the important precursors of volatile aroma compounds (Mottram, 1998). Therefore, the accumulation of amino acids during postmortem aging plays crucial roles in improving meat flavor.

| Degradation of ATP contributing to nucleotides accumulation during postmortem aging
Nucleotides mainly come from ATP metabolism in Tan lamb. ATP is the energy substance of metabolic reactions in both living animals and early postmortem muscles. The activation of glycolysis at the early stage of aging is to maintain ATP levels in postmortem Tan lamb muscles. However, due to the limited energy production capacity of glycolysis and intensified catabolism, the residual ATP was degraded. In the process of Tan lamb aging, nucleotides were degraded into adenosine diphosphate, adenosine, inosine, and hypoxanthine under the action of a series of enzymes. Nucleoside-diphosphate kinase was observed significantly increasing from day 0 to day 4, and decreasing from day 4 to day 8 (Table 2). Both inosine and hypoxanthine increased significantly during the whole period of aging (Table 3), which could be due the degradation of ATP into IMP and AMP, and further into inosine. The results of this study were consistent with previous research (Aliani et al., 2013). The GC-TOF-MS-based metabolomic was used in a recent study to investigate the taste-related metabolites in aged pork. The hypoxanthine was found increasing after 14-day aging (Tamura et al., 2022), which is consistent with the results of the present study. IMP, an important flavor substance giving umami taste in meat, has degradation F I G U R E 6 The regulative relationships between differentially abundant proteins and differential metabolites in Tan lamb in pair comparison during postmortem aging (a) The results of Group 4/0; (b) The results of Group 8/4. Note: The icon □ is for GO/KEGG terms, ○ for proteins/ genes, and ⌂ for metabolites, and the color green is for down-regulation, and red for up-regulation. The green line between icons indicates the inhibition between proteins and metabolites, red line indicates the activation between proteins and metabolites, and dotted lines indicate GO pathway and KEGG pathway products as important constituents in formation and development of meat flavor (Madruga et al., 2010). The bitterness in meat comes from hypoxanthine. Previously, it was found that the concentration of IMP dropped with a simultaneous increase in the concentrations of inosine, hypoxanthine, and ribose in beef with aging time (Tikk et al., 2006). The increase of inosine and hypoxanthine at day 4 and day 8 of aging in the present study is consistent with that in the previous study. Therefore, the mechanism of energy metabolism and ATP degradation in postmortem muscles is of great importance to meat flavor.

| CON CLUS IONS
In conclusion, the results of this study highlight the protein and metabolic changes occurring in Tan lamb muscles during postmortem aging. Muscle pH considerably decreased from day 0 to day 4, and increased from day 4 to day 8 (p < .05). The activity of LDH significantly increased from day 0 to day 4, and decreased from day 4 to day 8 (p < .05). Postmortem glycolysis was activated in 4 days, which directly affected variations in metabolic enzymes and triggered the accumulation of flavor-related carbohydrates. The free amino acids accumulated due to hydrolysis of structural proteins, with 3-hydroxy-L-proline, aspartic acid, and methionine increasing from day 0 to day 4, and aspartic acid, serine, threonine, tyrosine, phenylalanine, and D-phenylalanine from day 4 to day 8. The inosine and hypoxanthine accumulated due to the degradation of ATP.
The abundance of these water-soluble flavor precursors led to the change of meat flavor upon heating. Particularly, the structural proteins, actin (cytoplasmic 1), tropomyosin 2, Troponin C2 (fast skeletal type), actin alpha 1, and troponin T3 (fast skeletal type) could serve as the candidate predictors for monitoring of meat flavor precursor during postmortem storage.

This work was financially supported by the Innovation Research
Group Project of the National Natural Science Foundation of China [31860427].

CO N FLI C T O F I NTE R E S T S
All authors have declared no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.